This invention relates to methods for preparing and using Kluyveromyces for the production of polypeptides of interest which preferentially are secreted into the growth medium. The invention is exemplified by sequences useful in the production of chymosin and precursors thereof, tissue plasminogen activator (t-PA), and human serum albumin (HSA), in Kluyveromyces.
The bright promise of production of peptides in microorganisms has been tarnished by a number of factors. In many instances, where the peptide has been produced and retained in the cytoplasm, inclusion bodies have resulted requiring denaturation and renaturation of the protein, frequently with only partial or little success. In other instances, the peptide has been subjected to substantial degradation, so that not only are yields low, but also complicated mixtures are obtained which are difficult to separate. As a potential solution to these difficulties, the possibility of secretion of the desired peptide into the nutrient medium has been investigated. Secretion has met with limited success, since not all proteins have been found to be capable of secretion in the host which have been employed. Even when secreted, the processing of the peptide may result in a product which differs from the composition and/or conformation of the desired peptide. There is, therefore, substantial interest in being able to develop systems for the efficient and economic production of active peptides under conditions which allow for the use of the peptides in a wide variety of environments, both in vitro and in vivo.
European Patent Application (EPA) 0096430 discloses Kluyveromyces as a host for cloning and expression of foreign genes. However, no mention was made of the capability of secreting proteins into the growth medium.
The leader sequence of amyloglucosidase for Aspergillus is described by Boyle et al., EMBO J. (1984) 3:1581-1585 and Innis et al., Science (1985) 228:21-26. Lactase promoters are described by Bruenig et al., Nucleic Acids Res. (1984) 12:2327-2341. The use of signal peptides associated with mating-type xcex1-factor and of the enzymes invertase and acid phosphatase to direct the secretion of heterologous proteins in Saccharomyces has been described in EP-A-0123544 and by Smith et al., Science (1985) 229:1219.
Production of preprochymosin, prochymosin and chymosin in Saccharomyces has been studied by Mellor et al., Gene (1983) 24:1-14. When prochymosin is made intracellularly in Saccharomyces, only a low percentage of the prochymosin obtained is activatable. See Moir et al. in Developments in Industrial Biology (1985) 26:75-85; Mellor et al., Gene (1983) 24:1-14; Kingsman et al. in Biotechnology and Genetic Engineering Reviews Vol. 3 (1985) 376-418. The aggregated prochymosin produced by Saccharomyces required complicated methods of denaturation and renaturation to solubilize the prochymosin. See WO 83/04418 and EP-A-0114506.
Peptide production systems are provided comprising Kluyveromyces host strains, expression cassettes which include efficient transcriptional initiation and termination regions for use in Kluyveromyces and a gene, optionally containing a signal sequence for secretion, under the transcriptional and translational regulation of the regulatory regions. The cassettes are introduced into the Kluyveromyces host strain under conditions whereby the resulting transformants stably maintain the expression cassettes. Naturally occurring DNA and synthetic genes may be employed for the production of peptides of interest.